EP4348269A1 - Method to determine glyphosate and aminomethylphosphonic acid in a sample - Google Patents

Method to determine glyphosate and aminomethylphosphonic acid in a sample

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Publication number
EP4348269A1
EP4348269A1 EP22730525.7A EP22730525A EP4348269A1 EP 4348269 A1 EP4348269 A1 EP 4348269A1 EP 22730525 A EP22730525 A EP 22730525A EP 4348269 A1 EP4348269 A1 EP 4348269A1
Authority
EP
European Patent Office
Prior art keywords
solution
tosylate
glyphosate
aminomethylphosphonic acid
obtaining
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP22730525.7A
Other languages
German (de)
French (fr)
Inventor
Daniele MANCARDI
Silvia DE FRANCIA
Elisa ARRIGO
Sarah ALLEGRA
Francesco CHIARA
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Universita degli Studi di Torino
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Universita degli Studi di Torino
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Publication date
Application filed by Universita degli Studi di Torino filed Critical Universita degli Studi di Torino
Publication of EP4348269A1 publication Critical patent/EP4348269A1/en
Pending legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
    • G01N21/33—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using ultraviolet light
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2430/00—Assays, e.g. immunoassays or enzyme assays, involving synthetic organic compounds as analytes
    • G01N2430/20—Herbicides, e.g. DDT

Definitions

  • the present description concerns a new method to determine glyphosate and aminomethylphosphonic acid (AMPA) in organic and inorganic samples, and a kit thereof.
  • AMPA glyphosate and aminomethylphosphonic acid
  • N-(phosphonomethyl)glycine (glyphosate or GLYP) was first synthesised in 1974 and selected as herbicide for commercial distribution because of a selective inhibition of the shikimate pathways, a pivotal enzymes pathway supporting plant growth.
  • GLYP glyphosate
  • GLYP is transformed in aminomethylphosphonic acid (AMPA) by plants and in soil through oxidative deamination and further degradation to 2-methylphosphinicoacetic acid.
  • the object of this disclosure is to provide a new method for the determination with high accuracy of the presence or amount of GLYP and AMPA in an organic or inorganic sample, which overcomes the drawbacks of the known methods.
  • the present invention concerns a method of determining the presence or amount of glyphosate and aminomethylphosphonic acid in an organic or inorganic sample, the method comprising the following steps:
  • the present invention concerns a kit for performing the method of determining the presence or amount of glyphosate and aminomethylphosphonic acid in a sample as disclosed herein, wherein the kit comprises a high performance liquid chromatography reverse phase column, a vial containing a tosylchloride solution, vials containing standard solutions of glyphosate tosylate and aminomethylphosphonic acid tosylate and an instruction manual.
  • Figure 1 Graphical representation of the method of determining the presence or amount of glyphosate and aminomethylphosphonic acid as disclosed in the present application: (1) Mixing the sample with a tosylchloride solution in acetonitrile; (2) incubating the solution; (3) extracting with an organic aprotic solvent glyphosate tosylate and AMPA tosylate and subsequently washing the inferior phase with a washing solvent; (4) centrifuging, collecting the supernatant and drying; (5) reconstituting the dried supernatant with a mobile phase for HPLC-UV analysis; (6) HPLC-UV analysis.
  • FIG. 3 Time course of glyphosate degradation to AMPA.
  • AMPA and Glyphosate in Cell Culture Medium after 90min of incubation with spiked solution at lOOmg/ml of Glyphosate (point line); AMPA and Glyphosate in lysed cells after 90min of incubation with spiked solution at lOOmg/ml of Glyphosate in cell culture medium (dash line).
  • GLYP-based herbicides have been extensively used worldwide for the last 40 years and we are facing a continuous exposure to undetermined levels GLYP and AMPA because of the residues in water, soil, fruits and food in general.
  • the effects on human and animal health of GLYP and AMPA is still profoundly disputed. Nevertheless, the progress of the research about potential deleterious outcome needs to be supported by precise quantification of GLYP and AMPA in samples such as biological fluids, cellular and tissue specimens, food processing samples, among others.
  • samples such as biological fluids, cellular and tissue specimens, food processing samples, among others.
  • the method disclosed herein aims to unify protocols to determine GLYP and AMPA in different organic and inorganic samples, preferably biological samples, providing a kit for derivatization and analysis with UV chromatography.
  • the kit developed represents a practical resource for experimental, medical, quality control, alimentary and several other applications.
  • Most laboratories equipped with LC-MS/MS or HPLC-RF can perform determination of GLYP and AMPA with high cost and low reproducibility.
  • the present method will allow every laboratory endowed with a HPLC-UV system to extract and quantify the two molecules from any (organic or inorganic) sample with high reproducibility, amplifying the access to data about GLYP and AMPA concentrations .
  • the present invention concerns a method of determining the presence or amount of glyphosate and aminomethylphosphonic acid in an organic or inorganic sample, the method comprising the following steps:
  • the tosylchloride solution comprises tosylchloride and acetonitrile (100%), wherein tosylchloride is present in a concentration of 30.0-60.0 mM, preferably 50.0 mM.
  • the sample in step (i) is mixed with the tosylchloride solution and a buffer.
  • the buffer has a pH comprised between 10.0 and 12.0, preferably the pH is 11.0.
  • the buffer is selected from phosphate, bicarbonate, formate and acetate buffer.
  • the incubation of step (ii) is carried out at a temperature of 40-60 °C, preferably at 50 °C.
  • the incubation is carried out for a period of 2- 10 min., preferably 5 min..
  • the incubation is carried out in a thermostatically-controlled water bath.
  • GLYP and AMPA react with the tosylchloride to generate GLYP tosylate and AMPA tosylate as shown below in scheme 1, wherein panel A refers to GLYP and panel B refers to AMPA.
  • the tosylate derivatives of GLYP and AMPA are extremely useful during the analytical reactions because they are easily extracted from the sample (either organic or inorganic) according to the method described herein (namely, step (iii)), allowing an easy determination by UV spectrometry.
  • the organic aprotic solvent used in the extraction of step (iii) has a dielectric constant (8 r ) value comprised between 2.0 and 6.0.
  • the organic aprotic solvent contains at least one oxygen atom, that allows, thanks to its dipole- dipole forces, weak molecular interactions between solvent molecules and GLYP and AMPA.
  • the organic aprotic solvent is selected from ethyl acetate, isobutyl alcohol, dichloromethane, ethyl ether, methyl sulphide, propyl ether, isopropyl alcohol, toluene, chloroform, preferably ethyl acetate.
  • step (iii) is repeated twice.
  • step (iii) the centrifugation is carried out at a temperature of 2-10 °C, preferably at 5 °C. In one embodiment, the centrifugation is carried out for a period of time of 2-10 min, preferably 5 min.
  • the drying of the supernatant of step (iv) is carried out in a vacuum refrigerated concentrator.
  • the reconstituting solution in step (v) contains acetonitrile and water in a volume ratio comprised between 50:50 v/v and 75:25 v/v.
  • the reconstituting solution further contains a buffer.
  • the buffer has a pH comprised between 2.0 and 5.0, preferably the pH is 2.5.
  • the buffer is selected from formate, acetate, phosphate, citrate, trifluoroacetate, propionate.
  • step (v) the reconstituted supernatant is washed with a washing solvent capable of creating an azeotrope with the organic aprotic solvent used in step (iii) to remove water residues before proceeding to step (vi).
  • the washing solvent is selected from toluene, ethyl acetate, ethanol, benzene, methanol.
  • the organic aprotic solvent used in step (iii) is ethyl acetate then the washing solvent is toluene.
  • the chromatographic separation of step (vi) is carried out using a reverse phase column, preferably a C18 column, and a mobile phase.
  • the mobile phase comprises water and acetonitrile in volume ratios comprised between 90:10 v/v and 50:50 v/v.
  • the mobile phase further contains a buffer.
  • the buffer has a pH comprised between 2.0 and 4.0, preferably the pH is 3.0.
  • the buffer is selected from formate, acetate, phosphate, citrate, trifluoroacetate, propionate, preferably formate.
  • the detection by the UV- spectrometer of step (vii) is carried out at 240 nm.
  • the sample is an organic sample and is selected from cell culture medium, biological fluid, cell lysate and tissue lysate.
  • the sample is deproteinized and delipidized according to the common general knowledge (e.g. as disclosed in Zhu Jie Ping; Li Feng; Sheng Ye Shou, Medicinal Plant (2011);2(7):18-20) before carrying out step (i).
  • the present invention concerns a process for the preparation of standard solutions containing GLYP and AMPA tosylate to be used for the calibration of the method of determining the presence or amount of GLYP and AMPA as disclosed above.
  • the standard solutions of GLYP and AMPA tosylate contain GLYP and AMPA tosylate at a concentration comprised between 0.5 ug/ml and 20 ug/ml.
  • the process for the preparation of standard solutions containing GLYP and AMPA tosylate comprises the following steps: a) a GLYP or AMPA solution is mixed with a tosylchloride solution obtaining a first aqueous phase and a first organic phase; b) the first aqueous phase is rinsed with an aprotic solvent having a dielectric constant (s r ) value lower than 10, and brought to pH 1.5-2.5 obtaining a second aqueous phase and a second organic phase; c) the first and second organic phases are mixed together, anhydrified, and then mixed with the aprotic solvent, obtaining a third organic phase; d) the third organic phase is dried obtaining a powder; e) the powder is suspended in a further organic aprotic solvent having a dielectric constant (s r ) value comprised between 25 and 40 obtaining a solution; f) drying the solution; and g) resuspending the dried solution in the aprotic solvent obtaining a standard solution of G
  • the tosylchloride solution contains the organic aprotic solvent having a dielectric constant ( r ) value lower than 10.
  • the organic aprotic solvent has a dielectric constant (s r ) value comprised between 2.0 and 6.0.
  • the organic aprotic solvent is selected from ethyl acetate, isobutyl alcohol, dichloromethane, ethyl ether, methyl sulphide, propyl ether, isopropyl alcohol, toluene, chloroform, preferably ethyl acetate.
  • step b) is repeated twice.
  • the pH of the first aqueous phase is brought to pH 2.0, preferably adding 9M H2SO4.
  • the anhydrification operation in step c) is carried out using MgSCg.
  • the drying operation in step d) is carried out by nitrogen influx and further desiccation with a vacuum refrigerated concentrator.
  • the further organic aprotic solvent having a dielectric constant (c r ) value comprised between 25 and 40 is selected from methanol, acetone and acetonitrile .
  • GLYP and AMPA standard solutions are ready to be used, possibly operating one or more dilutions of the same to obtain the desired concentrations, in step (vi) of the method of determining GLYP and AMPA as disclosed herein.
  • the present invention concerns a kit for performing a method of determining the presence or amount of glyphosate and aminomethylphosphonic acid in a sample as disclosed herein, wherein the kit comprises: - a high performance liquid chromatography reverse phase column, a vial containing a derivatization solution comprising tosylchloride,
  • the standard solutions contain GLYP tosylate and AMPA tosylate in a concentration comprised between 0.5 ug/ml and 20.0 ug/ml.
  • the kit further contains in different vials at least one of: water and acetonitrile in a volume ratio 90:10 v/v and optionally a buffer having a pH comprised between 2.0 and 4.0; water and acetonitrile in a volume ratio 50:50 v/v and optionally a buffer having a pH comprised between 2.0 and 4.0; an organic aprotic solvent having a dielectric constant (s r ) value comprised between 25 and 40 (preferably methanol); acetonitrile .
  • s r dielectric constant
  • the experimental and protocol design has been a two-years process because of the continuous refining of the procedures in order to obtain a solid and novel method to extract and quantify glyphosate and its derivative AMPA obtained from several biological and inorganic matrices.
  • the inventors started investigating the biological effects of glyphosate and AMPA in late 2018, with an interdisciplinary team background, spanning from cardiotoxicity (both at tissue and cellular level) to clinical pharmacology. That being said, to the best of the inventors' knowledge, there is not a single publication and/or patent application about the extraction and detection of glyphosate and AMPA based on a HPLC-UV method coupled with a derivatization procedure comprising use of tosylchloride.
  • the extraction and quantification method disclosed herein is oriented toward UV detection with a widespread applications scenario.
  • Several reports refer to the tandem mass technique, which is a 2 nd level analysis (confirmation analysis), while the method disclosed herein can be widely applied in the quantification of spectrophotometric detectable analytes.
  • the UV analytical apparatus is very affordable.
  • the present invention clearly overcomes other critical issues often related to quantification of glyphosate and AMPA in biological matrices, that are due "...to the polar nature of the molecule" and "...to matrix interferences, direct injection methods are only applicable for relatively clean matrices, such as water or simple water-based matrices ".
  • the prior art methods use a C18-5 column with a 0.2 M phosphate buffer - acetonitrile 85/15 v/v as a mobile phase
  • step (iii) One of the characterizing features of the method disclosed herein is the development of an extraction step for the tosylate derivatives of GLYP and AMPA (step (iii)) which allows to greatly improve the reliability and reproducibility of the analytical detection of these analytes by HPLC-UV in an organic or inorganic sample.
  • This extraction step actually provides for a simpler procedure with an easy determination by UV spectrometry. Not all the analysis laboratories have mass detectors availability. Giving the possibility of using simpler and cheaper instrumentation can ensure greater applicability of our developed method.
  • the validation method used by the present inventors is based on higher level of samples required for the standardization of analytical methods: UNI EN ISO IEC 17025:2018.
  • the performance parameters have been tested with intra-laboratory and inter-laboratory analytical protocols as requested by par. 7.2.2. of UNI EN ISO IEC 17025:2018.
  • the parameters evaluated are: Range of linearity and normality test of Shapiro- Wilk;
  • LOD Limit Of Detection
  • LOQ Limit Of Quantification
  • IF Interval of Confidence
  • the inventors therefore, obtained a transversal extraction method applicable to all organic and inorganic samples with an optimisation of derivatising procedures and a thermodynamic control of the unimolecular nucleophilic substitution reaction (SN1) between GLYP or AMPA and 4-toluenesulfonyl chloride.
  • SN1 unimolecular nucleophilic substitution reaction
  • the kit for GLYP and AMPA extraction and quantification disclosed herein is based on a derivatization procedure which is completely novel and will allow operators to reduce costs and time to perform the assay, improving repeatability and standardization among laboratories.
  • the strengths of the present method are a high specificity for biological matrices (e.g. toxicological application) and an affordable analytical technique (HPLC-UV) applicable to a vast sample processing capacity.
  • the kit represents an inexpensive and robust tool for determining the presence or amount of GLYP and AMPA, with high specificity and easy to use.
  • the kit can be used in any laboratory, since regular laboratory supply (plasticware, pipets, centrifuge and similar) and HPLC- UV system (no column needed) are needed.
  • the method is suitable for toxicological monitoring of glyphosate and AMPA in cellular/tissue lysate and biological fluids (e.g. plasma and serum).
  • biological fluids e.g. plasma and serum.
  • the GLYP and AMPA tosylate standard solutions are prepared as disclosed below.
  • a. The GLYP (or AMPA) containing solution is mixed with a tosylchloride solution containing ethyl acetate obtaining a first aqueous phase and a first organic phase.
  • b. The obtained aqueous phase is rinsed twice with ethyl acetate and brought to pH 2 by adding 130 m ⁇ of 9M H2SO4, obtaining a second aqueous phase and a second organic phase.
  • c. The two organic phases are collected and mixed together, anhydrified and mixed with ethyl acetate obtaining a third organic phase.
  • the third organic phase is anhydrified with MgSCq obtaining a powder.
  • the powder is then suspended in ethyl acetate obtaining a solution.
  • the solution is dried through nitrogen influx and transferred for final exsiccation in a vacuum refrigerated concentrator obtaining a powder.
  • the powder is then suspended in 500 m ⁇ methanol and dried again through vacuum refrigerated concentration and subsequently resuspended in ethyl acetate .
  • Sample cell culture media, biological fluids (plasma, serum), cell and tissue lysates and inorganic samples
  • sample is a cellular or tissue lysate, it must be deproteinized and delipidized as disclosed in [Zhu Jie Ping; Li Feng; Sheng Ye Shou; Medicinal Plant (2011);2(7):18-20];
  • Solution A Aqueous buffer with pH range 2.0 - 3.5
  • Solution B Organic Solvent (dielectric constant between 25 - 40) with 10%v/v of Solution A; Volume injection: 5.Oul - 50.0ul
  • the calibration method is based on linear regression of standards injection.
  • the calibration curve is derived from standards with increasing dilution factors.
  • the chromatographic method is performing and the resolution of peaks is appreciable in Figure 3.
  • the point line represents the chromatographic signals of AMPA and GLYP in Cell Culture Medium after 90 min of incubation with spiked solution at 100 mg/ml of GLYP.
  • the dash line represents the chromatographic signals of AMPA and GLYP in lysed cells after 90 min of incubation with spiked solution at 100 mg/ml of GLYP in cell culture medium.
  • the concentration of AMPA and GLYP referred to Figure 3 are reported in Table 3.
  • the continuous line in chromatogram of Figure 3 represents the standard solution of AMPA and GLYP tosylates for peaks identification .
  • Resolution represents the column ability to separate peaks of interest. Resolution is based on efficiency (N), selectivity (a) and retention (k).
  • the selectivity (a) of a chromatographic method is intended as the ability to identify and quantify the analyte without deviation, or with the smallest possible deviation compared to the expected value when confounding compounds are present.
  • the estimated selectivity is performed by measures in complex mixture that are not normally in place when performing routing readings.
  • Specificity is the ability to identify and quantify the analyte with accuracy and precision.
  • the score of specificity is given by the measure of precision or value of accuracy (Bias%) with interfering molecules.

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Abstract

A method of determining the presence or amount of glyphosate and aminomethylphosphonic acid in a sample, the method comprising the following steps: (i) mixing the sample suspected of containing glyphosate and aminomethylphosphonic acid with a tosylchloride solution obtaining a first solution; (ii) incubating the first solution, thus obtaining glyphosate tosylate and aminomethylphosphonic acid tosylate; (iii) extracting glyphosate tosylate and aminomethylphosphonic acid tosylate from the first solution by:(a) mixing the first solution with an organic aprotic solvent having a dielectric constant lower than 10, (b) centrifuging and (c) collecting a supernatant; (iv) drying the supernatant; (v) reconstituting the supernatant in acetonitrile, obtaining a reconstituted supernatant; (vi) chromatographically separating glyphosate tosylate and aminomethylphosphonic acid tosylate from other constituents in the reconstituted supernatant by a high-performance liquid chromatography (HPLC) column; (vii) detecting by a UV- spectrometer the chromatographically separated glyphosate tosylate and aminomethylphosphonic acid tosylate to determine the presence or amount of glyphosate and aminomethylphosphonic acid contained in the sample.

Description

"Method to determine glyphosate and aminomethylphosphonic acid in a sample" kick
Field of the invention
The present description concerns a new method to determine glyphosate and aminomethylphosphonic acid (AMPA) in organic and inorganic samples, and a kit thereof.
Background art
N-(phosphonomethyl)glycine (glyphosate or GLYP) was first synthesised in 1974 and selected as herbicide for commercial distribution because of a selective inhibition of the shikimate pathways, a pivotal enzymes pathway supporting plant growth. Considered safe in terms of environmental impact and toxic effects on animals, with low production costs and high market demand, GLYP rapidly reached a worldwide diffusion for extensive agricultural use and it has been the most adopted herbicide over the last 40 years. GLYP is transformed in aminomethylphosphonic acid (AMPA) by plants and in soil through oxidative deamination and further degradation to 2-methylphosphinicoacetic acid.
Since the first introduction to the market, the effects of GLYP on plants have been largely investigated leading to the development of genetically modified crops, tolerant to the herbicide's action.
Except for occupational exposure to high levels of glyphosate-based herbicides, there were no early reports about toxicity in humans. Unlike fungi, plants, algae and bacteria, animals retrieve shikimate-derived aromatic amino acids through their diet and lack the target enzyme. Nevertheless, the demonstrated effects on microbiota can indirectly account for deleterious outcomes on mammalian physiological processes [Front. Behav. Neurosci. (2017), 11:146]. Scientific literature about the biological effects of GLYP on humans is still controversial and still misses consensus among research groups and certifying agencies. In this context, establishing a consistent and transversal way to extract, determine and quantify GLYP and AMPA in biological samples, is of pivotal importance to address potential pharmacodynamic and pharmacokinetics features of the two molecules, yet to be described. Consistently, there are no identified molecular target for glyphosate in mammalian cells, nor any mechanisms of toxicity have been exhaustively demonstrated.
In mid 1980s, several attempts to quantify GLYP and AMPA have been made because of frequent episodes of suicide through ingestion of glyphosate-based formulations. Most of the described procedures to determine concentrations, however, are extremely complicated, time-consuming, and do not provide acceptable quantification [Jpn. J. Legal Med., (1988),42:128] .
The first reliable attempts to quantify GLYP and AMPA residues in a biological matrix, are almost simultaneously and come from two Japanese university laboratories. Although the early development (1991), these studies describe a method for the determination of GLYP and AMPA through tosylated derivatives [J. Chromatogr. (1991), 566 (1):239-43; J. Chromatogr. (1991), 571 (1-2):324-330], but no extraction from biological matrices was performed and analysis were provided only for standards. The authors report a procedure for simultaneous quantification of GLYP and AMPA trough p-toluensulphonylchloride in alkaline solution but the procedure is time consuming and unprecise. In some cases, researchers' attempts to quantify GLYP and AMPA in serum showed a strong interference by matrix components that affected retention time and chromatograms [Journal of Chromatography 566 (1991) 239- 243]. In other studies, although limited to soil samples, FMOC has been used to derivatize GLYP leading to the formation of an adduct absorbing in the UV range [J. Chem. Educ. 95(1) (2018) 136-140].
Notwithstanding many methods for the detection of GLYP and AMPA have been developed in the past years, there is still need of a method that allows an easy and reliable quantification of GLYP and AMPA in a sample.
Summary of the invention
The object of this disclosure is to provide a new method for the determination with high accuracy of the presence or amount of GLYP and AMPA in an organic or inorganic sample, which overcomes the drawbacks of the known methods.
According to the invention, the above object is achieved thanks to the subject matter recalled specifically in the ensuing claims, which are understood as forming an integral part of this disclosure.
In one embodiment, the present invention concerns a method of determining the presence or amount of glyphosate and aminomethylphosphonic acid in an organic or inorganic sample, the method comprising the following steps:
(i) mixing the sample suspected of containing glyphosate and aminomethylphosphonic acid with a tosylchloride solution obtaining a first solution;
(ii) incubating the first solution, thus obtaining glyphosate tosylate and aminomethylphosphonic acid tosylate;
(iii) extracting glyphosate tosylate and aminomethylphosphonic acid tosylate from the first solution, by (a) mixing the first solution with an organic aprotic solvent having a dielectric constant (sr) value lower than 10, (b) centrifuging and (c) collecting a supernatant;
(iv) drying the supernatant;
(v) reconstituting the supernatant in acetonitrile, obtaining a reconstituted supernatant;
(vi) chromatographically separating glyphosate tosylate and aminomethylphosphonic acid tosylate from other constituents in the reconstituted supernatant by a high-performance liquid chromatography (HPLC) column; and
(vii) detecting by a UV-spectrometer the chromatographically separated glyphosate tosylate and aminomethylphosphonic acid tosylate to determine the presence or amount of glyphosate and aminomethylphosphonic acid contained in the sample.
In one embodiment, the present invention concerns a kit for performing the method of determining the presence or amount of glyphosate and aminomethylphosphonic acid in a sample as disclosed herein, wherein the kit comprises a high performance liquid chromatography reverse phase column, a vial containing a tosylchloride solution, vials containing standard solutions of glyphosate tosylate and aminomethylphosphonic acid tosylate and an instruction manual.
Brief description of the drawings
The invention will now be described in detail, purely by way of an illustrative and non-limiting example and, with reference to the accompanying drawings, wherein:
Figure 1: Graphical representation of the method of determining the presence or amount of glyphosate and aminomethylphosphonic acid as disclosed in the present application: (1) Mixing the sample with a tosylchloride solution in acetonitrile; (2) incubating the solution; (3) extracting with an organic aprotic solvent glyphosate tosylate and AMPA tosylate and subsequently washing the inferior phase with a washing solvent; (4) centrifuging, collecting the supernatant and drying; (5) reconstituting the dried supernatant with a mobile phase for HPLC-UV analysis; (6) HPLC-UV analysis.
Figure 2: Representative chromatograms of calibration curves: Standard Solutions with increasing concentration of glyphosate and AMPA (5, 10, 20 ppm). The first peak refers to AMPA (RT = 3.16min) while second peak is relative to Glyphosate (RT = 5.08min). The minor peaks are due to solvent background noise.
- Figure 3: Time course of glyphosate degradation to AMPA. AMPA and Glyphosate in Cell Culture Medium after 90min of incubation with spiked solution at lOOmg/ml of Glyphosate (point line); AMPA and Glyphosate in lysed cells after 90min of incubation with spiked solution at lOOmg/ml of Glyphosate in cell culture medium (dash line).
Detailed description of the invention
In the description that follows, numerous specific details are given to provide a thorough understanding of the embodiments. The embodiments can be practiced without one or more of the specific details, or with other methods, components, materials, etc. In other instances, well-known structures, materials, or operations are not shown or described in detail to avoid obscuring aspects of the embodiments.
Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.
The headings provided herein are for convenience only and do not interpret the scope or meaning of the embodiments.
GLYP-based herbicides have been extensively used worldwide for the last 40 years and we are facing a continuous exposure to undetermined levels GLYP and AMPA because of the residues in water, soil, fruits and food in general. The effects on human and animal health of GLYP and AMPA is still profoundly disputed. Nevertheless, the progress of the research about potential deleterious outcome needs to be supported by precise quantification of GLYP and AMPA in samples such as biological fluids, cellular and tissue specimens, food processing samples, among others. At today, there is no transversal protocol able to unify the quantification procedures to be adopted by R&D, quality control, University and other institutional laboratories.
The method disclosed herein aims to unify protocols to determine GLYP and AMPA in different organic and inorganic samples, preferably biological samples, providing a kit for derivatization and analysis with UV chromatography. The kit developed represents a practical resource for experimental, medical, quality control, alimentary and several other applications. At today, in fact, mostly laboratories equipped with LC-MS/MS or HPLC-RF can perform determination of GLYP and AMPA with high cost and low reproducibility. The present method will allow every laboratory endowed with a HPLC-UV system to extract and quantify the two molecules from any (organic or inorganic) sample with high reproducibility, amplifying the access to data about GLYP and AMPA concentrations .
In one embodiment, the present invention concerns a method of determining the presence or amount of glyphosate and aminomethylphosphonic acid in an organic or inorganic sample, the method comprising the following steps:
(i) adding to the sample suspected of containing glyphosate and aminomethylphosphonic acid a tosylchloride solution obtaining a first solution;
(ii) incubating the first solution, thus obtaining glyphosate tosylate and aminomethylphosphonic acid tosylate;
(iii) extracting glyphosate tosylate and aminomethylphosphonic acid tosylate from the first solution, by (a) mixing the first solution with an organic aprotic solvent having a dielectric constant (sr) value lower than 10, (b) centrifuging and (c) collecting a supernatant;
(iv) drying the supernatant;
(v) reconstituting the supernatant in acetonitrile, obtaining a reconstituted supernatant;
(vi) chromatographically separating glyphosate tosylate and aminomethylphosphonic acid tosylate from other constituents in the reconstituted supernatant by a high-performance liquid chromatography (HPLC) column; and
(vii) detecting by a UV-spectrometer the chromatographically separated glyphosate tosylate and aminomethylphosphonic acid tosylate to determine the presence or amount off glyphosate and aminomethylphosphonic acid contained in the sample.
The method object of the present application is graphically shown in Figure 1.
In one embodiment, the tosylchloride solution comprises tosylchloride and acetonitrile (100%), wherein tosylchloride is present in a concentration of 30.0-60.0 mM, preferably 50.0 mM.
In one embodiment, in step (i) the sample is mixed with the tosylchloride solution and a buffer. In one embodiment, the buffer has a pH comprised between 10.0 and 12.0, preferably the pH is 11.0. In one embodiment, the buffer is selected from phosphate, bicarbonate, formate and acetate buffer.
In one embodiment, the incubation of step (ii) is carried out at a temperature of 40-60 °C, preferably at 50 °C. The incubation is carried out for a period of 2- 10 min., preferably 5 min.. Preferably, the incubation is carried out in a thermostatically-controlled water bath.
During incubation, GLYP and AMPA react with the tosylchloride to generate GLYP tosylate and AMPA tosylate as shown below in scheme 1, wherein panel A refers to GLYP and panel B refers to AMPA.
The tosylate derivatives of GLYP and AMPA are extremely useful during the analytical reactions because they are easily extracted from the sample (either organic or inorganic) according to the method described herein (namely, step (iii)), allowing an easy determination by UV spectrometry.
D = 50°C
Scheme 1
In one embodiment, the organic aprotic solvent used in the extraction of step (iii) has a dielectric constant (8r) value comprised between 2.0 and 6.0. In one embodiment, the organic aprotic solvent contains at least one oxygen atom, that allows, thanks to its dipole- dipole forces, weak molecular interactions between solvent molecules and GLYP and AMPA. In one embodiment, the organic aprotic solvent is selected from ethyl acetate, isobutyl alcohol, dichloromethane, ethyl ether, methyl sulphide, propyl ether, isopropyl alcohol, toluene, chloroform, preferably ethyl acetate.
In one embodiment, the extraction of step (iii) is repeated twice.
In one embodiment, in step (iii) the centrifugation is carried out at a temperature of 2-10 °C, preferably at 5 °C. In one embodiment, the centrifugation is carried out for a period of time of 2-10 min, preferably 5 min.
In one embodiment, the drying of the supernatant of step (iv) is carried out in a vacuum refrigerated concentrator.
In one embodiment, in step (v) the reconstituting solution contains acetonitrile and water in a volume ratio comprised between 50:50 v/v and 75:25 v/v. In one embodiment, the reconstituting solution further contains a buffer. In one embodiment, the buffer has a pH comprised between 2.0 and 5.0, preferably the pH is 2.5. Preferably, the buffer is selected from formate, acetate, phosphate, citrate, trifluoroacetate, propionate.
In one embodiment, in step (v) the reconstituted supernatant is washed with a washing solvent capable of creating an azeotrope with the organic aprotic solvent used in step (iii) to remove water residues before proceeding to step (vi). In one embodiment, the washing solvent is selected from toluene, ethyl acetate, ethanol, benzene, methanol. For example, if the organic aprotic solvent used in step (iii) is ethyl acetate then the washing solvent is toluene.
In one embodiment, the chromatographic separation of step (vi) is carried out using a reverse phase column, preferably a C18 column, and a mobile phase. In one embodiment, the mobile phase comprises water and acetonitrile in volume ratios comprised between 90:10 v/v and 50:50 v/v. In one embodiment, the mobile phase further contains a buffer. In one embodiment, the buffer has a pH comprised between 2.0 and 4.0, preferably the pH is 3.0. The buffer is selected from formate, acetate, phosphate, citrate, trifluoroacetate, propionate, preferably formate. In one embodiment, the detection by the UV- spectrometer of step (vii) is carried out at 240 nm.
In one embodiment, the sample is an organic sample and is selected from cell culture medium, biological fluid, cell lysate and tissue lysate. When the sample is a cell or tissue lysate, the sample is deproteinized and delipidized according to the common general knowledge (e.g. as disclosed in Zhu Jie Ping; Li Feng; Sheng Ye Shou, Medicinal Plant (2011);2(7):18-20) before carrying out step (i).
In one embodiment, the present invention concerns a process for the preparation of standard solutions containing GLYP and AMPA tosylate to be used for the calibration of the method of determining the presence or amount of GLYP and AMPA as disclosed above. Preferably, the standard solutions of GLYP and AMPA tosylate contain GLYP and AMPA tosylate at a concentration comprised between 0.5 ug/ml and 20 ug/ml. The process for the preparation of standard solutions containing GLYP and AMPA tosylate comprises the following steps: a) a GLYP or AMPA solution is mixed with a tosylchloride solution obtaining a first aqueous phase and a first organic phase; b) the first aqueous phase is rinsed with an aprotic solvent having a dielectric constant (sr) value lower than 10, and brought to pH 1.5-2.5 obtaining a second aqueous phase and a second organic phase; c) the first and second organic phases are mixed together, anhydrified, and then mixed with the aprotic solvent, obtaining a third organic phase; d) the third organic phase is dried obtaining a powder; e) the powder is suspended in a further organic aprotic solvent having a dielectric constant (sr) value comprised between 25 and 40 obtaining a solution; f) drying the solution; and g) resuspending the dried solution in the aprotic solvent obtaining a standard solution of GLYP or AMPA tosylate.
In one embodiment, the tosylchloride solution contains the organic aprotic solvent having a dielectric constant (r) value lower than 10. In one embodiment, the organic aprotic solvent has a dielectric constant (sr) value comprised between 2.0 and 6.0. In one embodiment the organic aprotic solvent is selected from ethyl acetate, isobutyl alcohol, dichloromethane, ethyl ether, methyl sulphide, propyl ether, isopropyl alcohol, toluene, chloroform, preferably ethyl acetate.
In one embodiment, step b) is repeated twice. In one embodiment, the pH of the first aqueous phase is brought to pH 2.0, preferably adding 9M H2SO4.
In one embodiment, the anhydrification operation in step c) is carried out using MgSCg.
In one embodiment, the drying operation in step d) is carried out by nitrogen influx and further desiccation with a vacuum refrigerated concentrator.
In one embodiment, the further organic aprotic solvent having a dielectric constant (cr) value comprised between 25 and 40 is selected from methanol, acetone and acetonitrile .
The GLYP and AMPA standard solutions are ready to be used, possibly operating one or more dilutions of the same to obtain the desired concentrations, in step (vi) of the method of determining GLYP and AMPA as disclosed herein.
In one embodiment, the present invention concerns a kit for performing a method of determining the presence or amount of glyphosate and aminomethylphosphonic acid in a sample as disclosed herein, wherein the kit comprises: - a high performance liquid chromatography reverse phase column, a vial containing a derivatization solution comprising tosylchloride,
- a vial containing a standard solution of GLYP tosylate;
- a vial containing a standard solution of AMPA tosylate; and
- an instruction manual.
In one embodiment, the standard solutions contain GLYP tosylate and AMPA tosylate in a concentration comprised between 0.5 ug/ml and 20.0 ug/ml.
In one embodiment, the kit further contains in different vials at least one of: water and acetonitrile in a volume ratio 90:10 v/v and optionally a buffer having a pH comprised between 2.0 and 4.0; water and acetonitrile in a volume ratio 50:50 v/v and optionally a buffer having a pH comprised between 2.0 and 4.0; an organic aprotic solvent having a dielectric constant (sr) value comprised between 25 and 40 (preferably methanol); acetonitrile .
The experimental and protocol design has been a two-years process because of the continuous refining of the procedures in order to obtain a solid and novel method to extract and quantify glyphosate and its derivative AMPA obtained from several biological and inorganic matrices. The inventors started investigating the biological effects of glyphosate and AMPA in late 2018, with an interdisciplinary team background, spanning from cardiotoxicity (both at tissue and cellular level) to clinical pharmacology. That being said, to the best of the inventors' knowledge, there is not a single publication and/or patent application about the extraction and detection of glyphosate and AMPA based on a HPLC-UV method coupled with a derivatization procedure comprising use of tosylchloride. The extraction and quantification method disclosed herein is oriented toward UV detection with a widespread applications scenario. Several reports refer to the tandem mass technique, which is a 2nd level analysis (confirmation analysis), while the method disclosed herein can be widely applied in the quantification of spectrophotometric detectable analytes. The UV analytical apparatus is very affordable. The present invention clearly overcomes other critical issues often related to quantification of glyphosate and AMPA in biological matrices, that are due "...to the polar nature of the molecule..." and "...to matrix interferences, direct injection methods are only applicable for relatively clean matrices, such as water or simple water-based matrices ...". Some publications available in the literature, in fact, refer to FMOC derivatization techniques, which is more complex with respect to tosylchloride derivatization disclosed herein. Very often, the matrix effect in AMPA detection with MS/MS techniques is mitigated by employed Isotope internal standard: this approach is nevertheless very expensive and does not necessarily lead to reliable readings (Analytical Chemistry (2010);82(23):9671-9677). Some derivatization protocols based on FM0C-C1 lack accuracy when applied to biological matrices, while others are developed for fluorimetric detection.
With respect the detection methods employing tosylchloride for derivatizing GLYP and AMPA [J. Chromatogr. (1991), 566 (1):239-43; J. Chromatogr. (1991), 571(1-2):324-330], it is worth noting that such methods differ from the method disclosed herein in terms of:
- ratio of reaction, in terms of optimization of reaction volume of reactants to refine the reaction rate of the derivatization process;
- time of procedure (about 15 - 20 min compared to 5 min. of the present method);
- procedural steps (the derivatization solutions used in these prior art methods are not HPLC-UV injectables, therefore further processing is needed, contrary to what happens in the present method).
Moreover, the prior art methods use a C18-5 column with a 0.2 M phosphate buffer - acetonitrile 85/15 v/v as a mobile phase, while in the present method the mobile phase is an organic buffer vs. organic solvent (e.g. Solution A = lOmM Ammonium Formate in 90%v/v Acetonitrile and Solution B = 90%v/v Acetonitrile acidified with formic acid 0.1%v/v - Sol. A + Sol. B = Mobile phase) and can permit a higher resolution, allowing the achievement of an optimal pH for analytes separation.
One of the characterizing features of the method disclosed herein is the development of an extraction step for the tosylate derivatives of GLYP and AMPA (step (iii)) which allows to greatly improve the reliability and reproducibility of the analytical detection of these analytes by HPLC-UV in an organic or inorganic sample. This extraction step actually provides for a simpler procedure with an easy determination by UV spectrometry. Not all the analysis laboratories have mass detectors availability. Giving the possibility of using simpler and cheaper instrumentation can ensure greater applicability of our developed method.
The validation method used by the present inventors is based on higher level of samples required for the standardization of analytical methods: UNI EN ISO IEC 17025:2018. The performance parameters have been tested with intra-laboratory and inter-laboratory analytical protocols as requested by par. 7.2.2. of UNI EN ISO IEC 17025:2018. The parameters evaluated are: Range of linearity and normality test of Shapiro- Wilk;
LOD (Limit Of Detection), LOQ (Limit Of Quantification), IF (Interval of Confidence), repeatability and reproducibility;
Robustness;
Evaluation of uncertainty of measure with metrological approach and heuristic model of Horwitz.
The inventors, therefore, obtained a transversal extraction method applicable to all organic and inorganic samples with an optimisation of derivatising procedures and a thermodynamic control of the unimolecular nucleophilic substitution reaction (SN1) between GLYP or AMPA and 4-toluenesulfonyl chloride.
The kit for GLYP and AMPA extraction and quantification disclosed herein is based on a derivatization procedure which is completely novel and will allow operators to reduce costs and time to perform the assay, improving repeatability and standardization among laboratories. The strengths of the present method are a high specificity for biological matrices (e.g. toxicological application) and an affordable analytical technique (HPLC-UV) applicable to a vast sample processing capacity.
The kit represents an inexpensive and robust tool for determining the presence or amount of GLYP and AMPA, with high specificity and easy to use. The kit can be used in any laboratory, since regular laboratory supply (plasticware, pipets, centrifuge and similar) and HPLC- UV system (no column needed) are needed.
The technical innovations of the method disclosed herein are mainly:
Optimization of derivatization method (tosylchloride): less time, greater specificity, removal of interference; Optimization of chromatographic condition: less time, better separation of the analytic peaks and removal of chromatographic interferences, thus reducing matrix effect;
Applicability to biological systems: the method is suitable for toxicological monitoring of glyphosate and AMPA in cellular/tissue lysate and biological fluids (e.g. plasma and serum).
The technical innovations lead to several advantages in terms of:
Outstanding cost-effectiveness;
Easiness to apply;
Use of reagents and instruments largely available in laboratories.
Materials and Methods Standard Solutions Preparation
The GLYP and AMPA tosylate standard solutions are prepared as disclosed below. a. The GLYP (or AMPA) containing solution is mixed with a tosylchloride solution containing ethyl acetate obtaining a first aqueous phase and a first organic phase. b. The obtained aqueous phase is rinsed twice with ethyl acetate and brought to pH 2 by adding 130 mΐ of 9M H2SO4, obtaining a second aqueous phase and a second organic phase. c. The two organic phases are collected and mixed together, anhydrified and mixed with ethyl acetate obtaining a third organic phase. d. The third organic phase is anhydrified with MgSCq obtaining a powder. e. The powder is then suspended in ethyl acetate obtaining a solution. f. The solution is dried through nitrogen influx and transferred for final exsiccation in a vacuum refrigerated concentrator obtaining a powder. g. The powder is then suspended in 500 mΐ methanol and dried again through vacuum refrigerated concentration and subsequently resuspended in ethyl acetate .
Determination of GLYP and AMPA in a sample - steps i to v.
Reagents :
A. Derivatizing solution: 5mg of 4-Toluenesulfonyl chloride in 10ml of acetonitrile (HPLC grade)
B. Phosphate buffer 0.4M: 1:1 volume of 0.4M Na2HP04 and 0.4M Na3P04, pH 11.00
Sample: cell culture media, biological fluids (plasma, serum), cell and tissue lysates and inorganic samples
0. If the sample is a cellular or tissue lysate, it must be deproteinized and delipidized as disclosed in [Zhu Jie Ping; Li Feng; Sheng Ye Shou; Medicinal Plant (2011);2(7):18-20];
1. insert 200 ul of sample in a 2 ml tube and add 200 ul of reagent B and 200ul of reagent A, vortex 10 sec;
2. incubate the solution in thermostatic bath at 50°C for 5 min;
3. add 500 ul of ethyl acetate to the solution, mix vigorously for 5min then centrifuge at 5°C, 3500 RPM for 5 min. Collect the supernatant and transfer it in a new 2 ml tube. Repeat the procedure once with 300 ul ethyl acetate. Wash the supernatant with 150 ul of toluene in order to eliminate water residues;
4. dry the supernatant in a vacuum refrigerated concentrator; 5. reconstitute the dried supernatant in 200 ul of mobile phase (Phase A: Water + 0.1%v/v formic acid + 50mM Ammonium formate; Phase B: Acetonitrile :Water 9.5:0.5 + 0.1%v/v formic acid + 50mM Ammonium formate). At this point, the HPLC-UV analysis can be performed.
HPLC-UV analysis - steps vi and vii.
Column: Reversed Phase Core Shell (5miti, 4.6mmx250mm)
Elution: gradient with binary system (see Table 1) Solution A: Aqueous buffer with pH range 2.0 - 3.5 Solution B: Organic Solvent (dielectric constant between 25 - 40) with 10%v/v of Solution A; Volume injection: 5.Oul - 50.0ul
Detector Condition: kmax=240nm Flow: 1.0 ml/min
Table 1.
Calibration
The calibration method is based on linear regression of standards injection. The calibration curve is derived from standards with increasing dilution factors.
The typical chromatogram of calibration curve is reported in Fig.2, the retention time of peaks and relative concentration are reported respectively in Table 2 and in the legend of graph.
Table 2.
Results
The chromatographic method is performing and the resolution of peaks is appreciable in Figure 3. The point line represents the chromatographic signals of AMPA and GLYP in Cell Culture Medium after 90 min of incubation with spiked solution at 100 mg/ml of GLYP. The dash line represents the chromatographic signals of AMPA and GLYP in lysed cells after 90 min of incubation with spiked solution at 100 mg/ml of GLYP in cell culture medium. The concentration of AMPA and GLYP referred to Figure 3, are reported in Table 3. Ultimately the continuous line in chromatogram of Figure 3 represents the standard solution of AMPA and GLYP tosylates for peaks identification .
Table 3.
Comparative Example
In the following table 4 a comparison between the method of determining the amount of glyphosate and aminomethylphosphonic acid in a sample as disclosed in Kawai et al. (Journal Of Chromatography A 1991, 540:411- 415) and the method according to the present invention is provided.
Table 4 .
[1] Resolution represents the column ability to separate peaks of interest. Resolution is based on efficiency (N), selectivity (a) and retention (k).
[2] Repeatability, intended as precision and scored by RSD% (CV%) on a minimum of three different concentrations in 10 technical replicates.
[3] The selectivity (a) of a chromatographic method is intended as the ability to identify and quantify the analyte without deviation, or with the smallest possible deviation compared to the expected value when confounding compounds are present. The estimated selectivity is performed by measures in complex mixture that are not normally in place when performing routing readings.
[4] Specificity is the ability to identify and quantify the analyte with accuracy and precision. The score of specificity is given by the measure of precision or value of accuracy (Bias%) with interfering molecules.
[6] Time needed for the chromatographic separation.
[7] Limit of detection.
[8] Limit of quantification.
[9] Score of the accuracy of the analytical method.

Claims

Claims
1. A method of determining the presence or amount of glyphosate and aminomethylphosphonic acid in a sample, the method comprising the following steps:
(i) mixing the sample suspected of containing glyphosate and aminomethylphosphonic acid with a tosylchloride solution obtaining a first solution;
(ii) incubating the first solution, thus obtaining glyphosate tosylate and aminomethylphosphonic acid tosylate;
(iii) extracting glyphosate tosylate and aminomethylphosphonic acid tosylate from the first solution by: (a) mixing the first solution with an organic aprotic solvent having a dielectric constant lower than 10, (b) centrifuging and (c) collecting a supernatant;
(iv) drying the supernatant;
(v) reconstituting the supernatant in acetonitrile, obtaining a reconstituted supernatant;
(vi) chromatographically separating glyphosate tosylate and aminomethylphosphonic acid tosylate from other constituents in the reconstituted supernatant by a high-performance liquid chromatography (HPLC) column; (vii) detecting by a UV-spectrometer the chromatographically separated glyphosate tosylate and aminomethylphosphonic acid tosylate to determine the presence or amount of glyphosate and aminomethylphosphonic acid contained in the sample.
2. The method according to claim 1, wherein the tosylchloride solution comprises tosylchloride and acetonitrile and optionally a buffer solution having a pH comprised between 10.0 and 12.0.
3. The method according to any one of the preceding claims, wherein the incubation of step (ii) is carried out at a temperature of 40-60°C, preferably at 50 °C.
4. The method according to any one of the preceding claims, wherein the centrifugation of step (iii) is carried out at a temperature of 2-10°C.
5. The method according to any one of the preceding claims, wherein step (iii) is repeated twice.
6.The method according to any one of the preceding claims, wherein the drying of the supernatant of step (iv) is carried out in a vacuum refrigerated concentrator.
7.The method according to any one of the preceding claims, wherein the reconstitution of step (v) is carried out employing a reconstitution solution containing acetonitrile and water in a volume ratio comprised between 50:50 and 75:25 v/v, and optionally a buffer solution having a pH comprised between 2.0 and 5.0.
8.The method according to any one of the preceding claims, wherein in step (v) the reconstituted supernatant is washed with a washing solvent capable of creating an azeotrope with the organic aprotic solvent used in step (iii).
9. The method according to any one of the preceding claims, wherein the chromatographic separation of step (vi) is carried out using a reverse phase column.
10. The method according to any one of the preceding claims, wherein the chromatographic separation of step (vi) is carried out using a mobile phase comprising water and acetonitrile in a volume ratio comprised between 90:10 and 50:50 v/v, the mobile phase optionally containing a buffer having a pH comprised between 2.0 and 4.0.
11. The method according to any one of the preceding claims, wherein the detection by UV-spectrometer is carried out at 240 nm.
12. The method according to any one of the preceding claims, wherein the organic sample is selected from cell culture medium, biological fluid, cell lysate and tissue lysate.
13. A method of preparing standard solutions of GLYP tosylate and AMPA tosylate for carrying out the method of determining GLYP and AMPA in an organic or inorganic sample according to any one of the preceding claims, the method comprising the following steps:
(a) mixing a GLYP or AMPA solution with a tosylchloride solution, obtaining a first aqueous phase and a first organic phase;
(b) rinsing the first aqueous phase with an organic aprotic solvent having a dielectric constant value lower than 10, obtaining a second aqueous phase and a second organic phase;
(c) mixing the first and second organic phases together, anhydrifying, and adding the organic aprotic solvent, obtaining a third organic phase;
(d) drying the third organic phase obtaining a powder;
(e) suspending the powder in a further organic aprotic solvent having a dielectric constant value comprised between 25 and 40, obtaining a solution; (f) drying the solution;
(g) resuspending the dried solution in the organic aprotic solvent obtaining a standard solution of GLYP or AMPA tosylate.
14. Kit for performing a method of determining the presence or amount of glyphosate and aminomethylphosphonic acid in a sample according to any one of the preceding claims, wherein the kit comprises:
- a high performance liquid chromatography reverse phase column;
- a vial containing a tosylchloride solution;
- a vial containing a standard solution of GLYP tosylate;
- a vial containing a standard solution of AMPA tosylate; and
- an instruction manual.
15. The kit according to claim 14, further comprising at least one of: water and acetonitrile in a volume ratio 90:10 v/v and optionally a buffer having a pH comprised between 2.0 and 4.0; water and acetonitrile in a volume ratio 50:50 v/v and optionally a buffer having a pH comprised between 2.0 and 4.0; an organic aprotic solvent having a dielectric constant (eG) value comprised between 25 and 40; and acetonitrile.
EP22730525.7A 2021-05-27 2022-05-26 Method to determine glyphosate and aminomethylphosphonic acid in a sample Pending EP4348269A1 (en)

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